Wang CP, Lee YF, Chang C and Lee HJ - University of Rochester ...

Biochemical and Biophysical Research Communications 351 (2006) 204–208BBRCwww.elsevier.com/locate/ybbrcTransactivation of the proximal promoter of human oxytocin geneby TR4 orphan receptorChih-Pin Wang a,1 , Yi-Fen Lee b,1 , Chawnshang Chang b , Han-Jung Lee a, *a Department of Life Science, National Dong Hwa University, Hualien 97401, Taiwanb George Whipple Laboratory for Cancer Research, Department of Urology, University of Rochester Medical Center, Rochester, NY 14642, USAReceived 29 September 2006Available online 12 October 2006AbstractThe human testicular receptor 4 (TR4) shares structural homology with members of the nuclear receptor superfamily. Some othermembers of this superfamily were able to regulate the transcriptional activity of the human oxytocin (OXT) promoter by binding tothe first DR0 regulatory site. However, little investigation was conducted systematically in the study of the second dDR4 site ofOXT proximal promoter, and the relationship between the first and the second sites of OXT promoter. Here, we demonstrated forthe first time that TR4 could increase the proximal promoter activity of the human OXT gene via DR0, dDR4, and OXT (both DR0and dDR4) elements, respectively. TR4 might induce OXT gene expression through the OXT element in a dose-dependent manner. However,there is no synergistic effect between DR0 and dDR4 elements during TR4 transactivation. Taken together, these results suggestedthat TR4 should be one of important regulators of OXT gene expression.Ó 2006 Elsevier Inc. All rights reserved.Keywords: Oxytocin; Nuclear receptor superfamily; Orphan receptor; Gene regulationThe function of steroid and thyroid hormones is mediatedthrough the action of specific nuclear receptors [1].Members of the nuclear receptor superfamily are ligand-dependenttranscription factors that regulate the expressionof target genes by binding to their hormone response elements(HREs) [2]. Orphan receptors comprise a hugegroup of nuclear receptor-like proteins whose cognateligands remain to be identified. The human testicular receptor4 (TR4) (classified as NR2C2) is an orphan member ofthe nuclear receptor superfamily [3], that was initially isolatedfrom human prostate and testis cDNA libraries [4].TR4 directly modulates target gene expression throughbinding to HRE with consensus AGGTCA direct-repeat(DR). In general, the expression of TR4 transcripts occursin many mouse tissues. TR4 transcripts were detected as* Corresponding author. Fax: +886 3 8630340.E-mail address: hjlee@mail.ndhu.edu.tw (H.-J. Lee).1 These authors contributed equally to this work.early as embryonic day 9 (E9) in mouse embryos [5].TR4 transcripts were expressed highly in the central nervoussystem at E14.5–19.5 in rat [6], and in a stage-dependentand androgen-inductive manner in testis of rat [7].Study of TR4-knockout (TR4KO) mice further revealedthat TR4 might function as a master regulator in many signalingpathways, including female reproductive functionand maternal behavior [8].Oxytocin (OXT) is a multifunctional hormone that mediatesuterine contractions, milk ejection, specific behavioralpatterns, and natriuresis [9]. This peptide hormone containsnine amino-acid residues and is synthesized in vivo as part ofa long polypeptide precursor. The OXT gene has a characteristicpromoter, including overlapping elements for severalmembers of the nuclear receptor superfamily [10]. Basically,there are two regulatory footprints among the proximalOXT promoter, the first site is 164 to 145 upstream fromthe transcription initiation site, and the second one is 110 to79 [9]. Estrogen receptors (ERa and ERb) have been shownto bind to a palindromic estrogen response element (ERE) at0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.bbrc.2006.10.021

C.-P. Wang et al. / Biochemical and Biophysical Research Communications 351 (2006) 204–208 205the first site of the proximal promoter of the human OXTgene. This palindromic ERE overlaps with a directAGGTCA repeat, corresponding to a natural variant of aso-called DR0 (direct-repeat spaced by 0 nucleotide). Thesecond regulatory site contains three copies of AGGTCArepeat spaced by four nucleotides each, corresponding to adouble-DR4 (dDR4).Materials and methodsPlasmid constructions. pCMX-TR4 and pET14b-TR4 were describedpreviously [11,12]. pGL3-OXT-DR0 plasmid containing one copy of theDR0 element was generated by insertion of two annealing primers OXT-DR0U (5 0 -GATCGGTGACCTTGACCCCG-3 0 ) and OXT-DR0D (5 0 -GATCCGGGGTCAAGGTCACC-3 0 ) at BglII site of pGL3-Promotervector (Promega). pGL3-OXT-dDR4 plasmid consisting of one copy ofthe dDR4 element was constructed by insertion of two annealing primersOXT-dDR4U (5 0 -GATCCCTGACCCACGGCGACCCTCTGTGACCAAT-3 0 ) and OXT-dDR4D (5 0 -GATCATTGGTCACAGAGGGTCGCCGTGGGTCAGG-3 0 ) at BglII site of pGL3-Promoter. pGL3-OXTplasmid containing one copy of the OXT element (including both DR0and dDR4 elements) was generated by insertion of two annealing longprimers OXT-U (5 0 -GATCTGACCTTGACCCCGCCTGACCCACGGCGACCCTCTGTGACCA-3 0 ) and OXT-D (5 0 -GATCTGGTCACAGAGGGTCGCCGTGGGTCAGGCGGGGTCAAGGTCA-3 0 )atBglIIsite of pGL3-Promoter. All plasmids were sequenced by the ABI PRISM3100-Avant Genetic Analyzer (Applied Biosystems). DNA plasmid waspurified with the Nucleobond AX-100 Kit (Macherey-Nagel) according tothe manufacturer’s protocol.Western blot analysis. TR4 protein was overexpressed from pET14b-TR4-transformed Escherichia coli and purified with His-Bind column aspreviously described [13–15]. Western blot analysis was employed asdescribed previously [16]. Briefly, 1 lg of TR4 protein was separated bySDS–PAGE on a 12% polyacrylamide gel and electroblotted onto aPVDF membrane (Amersham) according to the manufacturer’s protocol.After nonspecific blocking, the membrane was treated with rabbit anti-6his antibody (Bethyl), followed by HRP-conjugated goat anti-rabbit Igantibody (MDBio, Taiwan), and then visualized by an enhanced chemiluminescencedetection kit (ECL Plus Western Blotting Detection System,Amersham).Electrophoretic mobility shift assay (EMSA). This assay was conductedmainly as previously described [17]. TR4 protein was generatedfrom a coupled in vitro transcription and translation system as previouslydescribed [11,17]. A 42 bp double-stranded oligonucleotide OXT probewas generated by annealing of primers OXT-U and OXT-D and digoxigenin(DIG)-labeled (Roche) according to the manufacturer’s protocol.Transient transfection and dual-luciferase assays. Human lung cancerA549 cells (American Type Culture Collection, CCL-185) were cultured aspreviously described [14]. Transient transfection and dual-luciferase assayswere conducted as previously described [12,18,19].ResultsEvolutionary sequence conservation among promoters ofOXT genesTwo regulatory footprints of the human OXT promoterare located between 167 and 149 (the first site, correspondingto nucleotide numbers 215 and 233 of AccessionNo. M11186, GenBank), and between 105 and 76 (thesecond site) (Fig. 1A). In the first site, a DR0 element isbetween nucleotide numbers 220 and 231, overlapping witha palindromic ERE. Consensus sequences of the DR0 elementin rat, mouse, and bovine showed 100, 100, and91.7% homologies with that of human, respectively. Inthe second site, a dDR4 element contains three copies ofAGGTCA consensus motif spaced by four nucleotideseach in human (nucleotide numbers 279 and 304) andbovine promoters, while one nucleotide deleted in a spaceregion in rat and mouse promoters. Consensus sequencesof dDR4 in rat, mouse, and bovine displayed 88.9, 83.3,and 88.9% homologies with that of human, respectively.These results suggested that OXT elements (includingDR0 and dDR4 elements) were evolutionally conservedamong these species.TR4 orphan receptor specifically binds to the human OXTproximal promoterTR4 was purified from cellular extracts isolated fromE. coli BL21(DE3) transformed with pET14b-TR4 expressingplasmid. TR4 protein was confirmed on a 12%SDS–PAGE, followed by Western blot analysis (Fig. 1B).TR4 protein corresponding to the expected molecular massof 67.3 kDa could be detected in both isopropyl-b-D-thiogalactopyranoside(IPTG)-induced cellular lysates (lane 2)and eluent after purification (lane 3), but not in non-inducedlysates (lane 1).A 42 bp double-stranded oligonucleotide OXT probewas generated by annealing two primers OXT-U andOXT-D, and DIG-labeled. TR4 protein was generatedfrom a coupled in vitro transcription and translation systemwith pET14b-TR4 plasmid. A specific DNA–protein complexwas revealed when TR4 was incubated with the OXTelement probe (Fig. 1C, lane 3). This DNA–protein complexcould be abolished in the presence of 100-fold molarexcess of unlabeled oligonucleotides (lane 4). NonspecificDNA–protein complexes appeared at the position rightbelow the specific complex in the mock-translated product(lane 2). These data indicated that TR4 could specificallybind to the OXT element of the human OXT proximal promoter.In addition, TR4 might also form specific DNA–protein complexes with DR0 and dDR4 elements in theEMSA, respectively (data not shown).TR4 activates OXT gene expression via DR0 and dDR4elementsTo investigate, whether TR4 could regulate the humanOXT gene expression through the interaction with proximalpromoter, we performed dual-luciferase assay inhuman A549 cells. Cells were co-transfected with variousluciferase reporter plasmids (Fig. 2) in the presence orabsence of pCMX-TR4 expression plasmid. In the case ofDR0 and dDR4 elements, TR4 was able to activate luciferasereporter gene activities of the pGL3-OXT-DR0(Fig. 3A) and pGL3-OXT-dDR4 (Fig. 3B) plasmids,respectively. These results suggested that TR4 might induceOXT gene expression through DR0 and dDR4 elements inproximal promoter, respectively.

206 C.-P. Wang et al. / Biochemical and Biophysical Research Communications 351 (2006) 204–208Fig. 1. Interaction between TR4 and OXT promoter. (A) Sequences of the proximal OXT promoters. Identical sequence is shown in capital, but differencefrom the human sequence is indicated in lower-case. Nucleotide numbers from different species with the Accession Numbers (GenBank) are indicated,while minus numbers denote nucleotides before the transcription initiation site in human. Potential AGGTCA repeats are indicated by arrows undersequences, and an ERE is shown by arrows above sequences. (B) Western blot analysis of TR4. Protein samples were extracted either from non-inducedE. coli strain BL21 (DE3) (lane 1), IPTG-induced E. coli (lane 2), or after purification (lane 3). Standard protein molecular masses are displayed on the left.TR4 protein, corresponding to the expected molecular mass of 67.3 kDa is indicated by an arrowhead. (C) Binding of the in vitro expressed TR4 to theOXT element. Lane 1 displays the DIG-labeled OXT probe alone. Binding reaction mixtures were incubated with the probe and either the mock-translatedproduct (lane 2) or the in vitro synthesized TR4 (lanes 3 and 4) in the presence of 100-fold molar excesses of unlabeled OXT (lane 4). The retardedcomplexes are indicated by an arrowhead for specific DNA–protein complexes, whereas nonspecific complexes appear below.The induction activity of OXT promoter by TR4 in a dosedependentfashionTo determine any synergistic effect between DR0 anddDR4 elements in the human OXT proximal promoter,A549 cells were transiently co-transfected with eitherpGL3-Promoter or pGL3-OXT reporter plasmid in thepresence or absence of pCMX-TR4 plasmid. In the caseof the OXT element, TR4 could regularly activate luciferasereporter gene activity of the pGL3-OXT plasmid(Fig. 4A). These data indicated that TR4 might induceOXT gene expression through the OXT element withoutsynergistic effect between DR0 and dDR4 elements.To further reveal this transactivation of OXT proximalpromoter by TR4, cells were co-transfected with pGL3-OXT reporter plasmid and increasing amounts ofpCMX-TR4 plasmid (Fig. 4B). Results showed that TR4could trigger the luciferase reporter activity via OXT proximalpromoter in a dose-dependent manner.DiscussionIn recent years, several reports have focused on the studyof the multiple HREs at the first site of the proximal promotersof human or rat OXT genes. These studies revealedthat this multiple HRE is responsive to estrogen, thyroidhormone, or retinoids [10]. In addition, previous study indicatedthat two orphan receptors, steroidogenic factor 1(SF-1) and chicken ovalbumin upstream promoter transcriptionfactor (COUP-TF), preferentially bound to thisDR0 element in the bovine OXT promoter [20]. Thesetwo orphan receptors appeared to compete for binding tothis DR0 element, since SF-1 was associated with OXT geneactivation and COUP-TF with its repression. Subsequently,

C.-P. Wang et al. / Biochemical and Biophysical Research Communications 351 (2006) 204–208 207A1210*intensityrelative86420pGL3 pGL3+TR4 pGL3-DR0 pGL3-DR0 +TR4B76*intensity54Fig. 2. Schematic structure of reporter gene plasmids. pGL3-OXT-DR0,pGL3-OXT-dDR4, and pGL3-OXT plasmids contain one copy of DR0,dDR4, and the full-length regulatory OXT elements in the senseorientation at the multiple cloning site (MCS) in front of the simian virus40 (SV40) promoter and the modified coding region of luciferase reportergene (Luc + ) in the parent pGL3-Promoter vector, respectively.relative3210pGL3 pGL3 + TR4 pGL3-dDR4 pGL3-dDR4 +TR4Fig. 3. TR4 induction of luciferase reporter gene activity of the OXTproximal promoter via DR0 and dDR4 elements, respectively. (A)Functional assay with pGL3-OXT-DR0 plasmid. (B) Functional assaywith pGL3-OXT-dDR4 plasmid. Human A549 cells were transiently cotransfectedwith different pGL3 reporter plasmids (2 lg of each) and 1 lgphRL-TK internal control plasmid in the presence or absence of 2.5 lgpCMX-TR4 expressing plasmid. All firefly luciferase activities werenormalized with Renilla luciferase activities. Data represent means ± SDof three independent experiments performed in duplicate. Significantdifference (P < 0.05) between control and experimental groups is markedwith an asterisk.two COUP members (COUP-TFII and Ear-2) were identifiedto suppress basal OXT promoter activity via DR0 anddDR4 elements [9]. Especially, the dDR4 element wasessential for this silencing effect. The specific bindingpreference to the DR0 element was in the order ofCOUP-TF = TRa > SF-1 > RAR > ER [10]. Nevertheless,the complex formed between the breast cell nuclear extractsand the DR0 element exposed that an unknown transcriptionfactor remained to be identified. This factor was noneof identified nuclear receptors, but was likely to be anothermember of the nuclear receptor superfamily [10].In the present study, we found that TR4 could directlybind to DR0, dDR4, and OXT elements, respectively.The TR4-DR0 binding complex could be abolished in thepresence of 100-fold molar excesses of unlabeled oligonucleotides,while the TR4-dDR4 complex might be impairedin the presence of only 30-fold molar excesses of competitors(data not shown). These data suggested that theDR0 element could be essential for mediating TR4 transactivationamong two regulatory sites. That is probably truedue to the consequence that TR4 possesses higher affinityto the DR0 element than the dDR4 element. Characterizationof the binding preference of TR4 to synthetic DRsindicated in the order of DR6 > DR5 > DR1 > DR3 >DR2 > DR4 [12]. It is interesting to note that the dDR4element of the human OXT promoter may represent anothertype of two direct-repeats spaced by 14 nucleotides apart(DR14). The medium-chain ethyl coenzyme A dehydrogenasegene promoter contains a complex regulatory elementwith four hexamer binding sites of nuclear receptors [21].This regulatory element previously conferred transcriptionalregulation by ARA/RXR and HNF-4 orphan receptor.COUP family homodimers were able to interact and functionwith an everted repeat with the same spacing of 14nucleotides (ER14) of this gene promoter.OXT is produced in neurons of the paraventricular andsupraoptic nuclei of the hypothalamus, as well as in specifictissues and cell types peripherally [22]. Physiological functionsof OXT have been well studied, and this peptide hormoneis known to affect both central and peripheralsystems, as well as behavior. In order to prove OXT mightbe a TR4 target gene, more direct physiological evidencemay come from the study of TR4KO mice [8]. Results demonstratedhigh rates of early postnatal mortality and significantgrowth retardation in TR4KO mice. TR4KO femalesfurther showed defects in reproduction and maternalbehavior. Especially, the reduced OXT expression in the

208 C.-P. Wang et al. / Biochemical and Biophysical Research Communications 351 (2006) 204–208AintensityrelativeBintensityrelative1086420121086420hypothalamic regions of TR4KO mice [8] suggested thatthe defect in regulatory levels of this peptide hormone,resulting from the disruption of TR4 expression.AcknowledgmentsWe are grateful to Drs. Ming-Huan Chan and Jyh-Ching Chou for critical reading and editing of the manuscript.This work was supported by the National ScienceCouncil, Taiwan.ReferencespGL3 pGL3 + TR4 pGL3-OXT pGL3-OXT +TR40 0.25 0.5 1 2 4 6amount of TR4 (microgram)Fig. 4. TR4 Induction of luciferase reporter gene activity of the OXTproximal promoter via the OXT element in a dose-dependent manner. (A)Functional assay with pGL3-OXT plasmid. (B) Functional assay ofdosage effect with pGL3-OXT plasmid. 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